During collagen synthesis by fibroblasts, cell-mediated assembly of collagen fibrils takes place in cell-surface crypts (FIG. 1A). The crypts are typically located on one pole of the synthetically active cell. Matrix components, including collagen are transported to the crypts, assembled and secreted into the extracellular space by vectorial discharge (FIG. 1B).
Within the crypts, individual procollagen molecules are prepared for self-assembly by enzymatic cleavage of their N- and C-terminal propeptides. Once these are cleaved, the remaining tropocollagen monomer (˜300 nm in length) will spontaneously self-assemble into secondary aggregate polymeric fibrils, which display the classic quarter-stagger arrangement. In vivo, the temperature and pH in the crypt are conducive to self-assembly. Thus, the rate of fibril formation is then likely to be dependent on the transport of procollagen to the crypt.
Typically, engineered connective tissue constructs are initiated by seeding fibroblast cells into a self-assembled collagen gel. However, such constructs typically do not contain collagen fibrils with sufficient order and typically do not have the load-bearing capacity of biological tissues.
There are few devices designed to construct collagen arrays that are ordered on the nanoscale. However, there have been some attempts, which are described below.
In one approach, some early researchers attempted to control collagen fibril orientation during fibrillogenesis by applying a large magnetic field to the vessel in which assembly was taking place (Guido et al., 1993). However, using a bulk magnetic field to align collagen fibrils during fibrillogenesis does not produce a high degree of alignment, nor does it allow control of orientation within the bulk collagen matrix that is generated.
In a more recent patent application, Braithwaite and Ruberti describe a method by which collagen fibrils can be aligned in sheets within a thin shear film (US patent application 20030141618). Alignment of the collagen fibrils is excellent, but the method is difficult to use due to the instability of the thin shear film and the problems associated with constructing multiple layers to produce a three-dimensional template. In addition, the constraint of assembly in a shearing flow places limitations on the ability to transport and allow binding of additional components during the self-assembly of the fibrils.
Braithwaite and Ruberti also describe a collagen nanoloom device. This device uses microfluidic driving forces to bring together the reacting molecules. In this system, collagen fibrils might form but would be uncontrollably driven from the nanoreactors by the exiting fluid flow. Further, the device, as depicted, draws fibrils from the reactors by moving away from a surface (to which formed collagen fibrils have adhered). Such a design would require that the “loom” always be tethered directly to this surface by a straight line of extruded collagen, which limits the ability of the system to produce patterns.
Finally, Wilson et al., 2001, describe a method of precisely placing collagen onto a substrate using dip pen nanolithography. This method requires the use of an atomic force microscope (AFM) type head and would require massive modification to produce large arrays of fibrils in three dimensions.
Thus, there remains a need for a system of devices and corresponding methods capable of producing two- and three-dimensional arrays of collagen fibrils and other polymer strands in a desired pattern. Such patterned arrays are important components for producing artificial cornea, tendon, bone, and other tissues and structures, particularly those involving extracellular matrix proteins.